Use of 2,3-dihydro-1-oxo-1h-pyrrolo(3,4-b)-quinoxaline-4,9-dioxides as animal growth promotants

ABSTRACT

3-HALOMETHYL-2-QUINOXALINECARBOXYLIC ACID, ALKYL ESTER 1,4-DIOXIDES USEFUL AS ANTIBACTERIAL AGENTS ARE CONVERTED, ON TREATMENT WITH AMMONIA AND AMINES TO 2,3-DIHYDRO-1OXO-1H-PYRROLO(3,4-B)QUINOXALINE-4,9-DIOXIDES ALSO USEFUL AS CHEMOTHERAPEUTIC AGENTS AND IN PROMOTING GROWTH AND IMPROVING FEED EFFECIENCY OF ANIMALS, IN GENERAL.

US. Cl. 424-250 8 Claims ABSTRACT OF THE DISCLOSURE 3-halomethyl-2-quinoxalinecarboxylic acid, alkyl ester 1,4-dioxides useful as antibacterial agents are converted, on treatment with ammonia and amines to 2,3-dihydro-1- oxo-lH-pyrrolo[3,4-b]quinoxaline-4,9-dioxides also useful as chemotherapeutic agents and in promoting growth and improving feed efliciency of animals, in general.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional copending application Ser. No. 25,543, filed Apr. 3, 1970.

BACKGROUND OF THE INVENTION This invention relates to quinoxaline-di-N-oxides, and more particularly to a unique series of 3-halomethyl-2- quinoxalinecarboxylic acid, alkyl ester 1,4-dioxides, useful as antibacterial agents and intermediates, and to 2,3-dihydro-1-oxo-1H-pyrrolo[3,4-b]quinoXaline-4,9-dioxides, the cyclization products of said halomethyl esters with ammonia and amines, also possessing antibacterial activity against pathogenic micro-organisms andto methods for the promotion of weight gain and food consumption in animals.

Continuing efforts to discover new and more useful antibacterial agents have led, over the years, to the development of a wide variety of prototype organic compounds including numerous congeners of quinoxaline-di-N-oxides. Landquist et al., J. Chem. 'Soc., 2052 (1956), in a search for compounds of improved antibacterial or antiprotozoal activity, reported the preparation of several derivatives of Z-methyland 2,3-dimethyl-quinoxaline-di-N-oxides in which the methyl groups were converted to groups such as bisbromomethyl-, acetoxymethyland hydroxymethyl including 3 methyl-Z-carbethoxyquinoxaline-di-N-oxide. However, no utility is alleged for any of these compounds. French Pat. M3,717, granted I an. 3, 1966, discloses 2-quinoxalinecarboxamide-di-N-oxides in which the carboxamide group may be substituted with an alkyl, substituted alkyl, aryl, cycloalkyl, aralkyl, or cycloalkyl group.

Belgian Pat. 697,976, granted Nov. 3, 1967, describes a variety of N-substituted derivatives of 3-methy1-2-quinoxalinecarboxamide-di-N-oxide in which the N-substituent is phenyl, substituted phenyl, dodecyl or ethyl, as well as the corresponding 3-methyl-2-earbethoxyquinoxaline-di- N-oxide. They are said to be of value as intermediates for United States Patent O J ill, CII Y 3,773,950 Patented Nov. 20, 1973 the preparation of vegetation protection agents and pharmaceutical agents. Belgian Pats. 721,724; 721,725; 721,- 727 and 721,728; published Apr. 2, 1969, describe a variety of N-substituted 3-methy1-2-quinoxalinecarboxamide-di-N-oxide derivatives wherein the N-substituent is a hydroxyalkyl, lower alkoxyalkyl, carboalkoxyalkyl, monoalkylaminoalkyl or di(alkyl)aminoal=kyl group as antibacterial agents, and include 3-chloromethyland 3-bromomethyl-2-quinoxalinecarboxamide-1,4-dioxides.

SUMMARY 'OF THE INVENTION The novel antibacterial and growth promotant quinoxaline-di-N-oxides of this invention are represented by the formulae to co R N 0 a 9 I 7 and X N-R wherein X is a substituent at the 6- or 7-position consisting of hydrogen, fluorine, chlorine, bromine, tri-fiuoromethyl, methyl or methoxy;

R is alkyl containing from 1 to 3 carbon atoms;

R, is hydrogen or alkyl containing from 1 to 3 carbon atoms; and

Y is bromine or chlorine.

Of particular interest, because of their in vitro antibacterial activity and as intermediates, leading to congeners of Formula II, are compounds of Formula I wherein R is alkyl containing up to 3 carbon atoms, X is hydrogen and Y is chlorine or bromine, and of Formula II wherein X is hydrogen and R is hydrogen or methyl. Compounds of particular interest because of their activity as in vitro and in vivo antibacterial agents, as well as animal growth promotant activity are those of Formula 11 wherein X is hydrogen and R is hydrogen or methyl.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the process employed for preparing the 3-halomethyl-2-quinoxalinecarboxylic acid, alkyl esters of the present invention, an appropriately substituted 3-methyl-2-quinoxalinecarboxylic acid, alkyl ester 1,4-dioxide is halogenated employing molecular bromine or chlorine.

In practice, compounds of Formula I are prepared by adding an equimolar amount, plus as much as a 10% excess, of the halogenating agent, dissolved in a reactioninert solvent such as methylene chloride, or chloroform, to the requisite 3-methyl-2-quinoxalinecarboxylic acid, alkyl ester 1,4-dioxide dissolved in a highly polar, water miscible reaction-inert solvent, e.g., dimethylformamide. It is preferred that the reaction mixture be preheated from -100 C. before the additionlof the halogenating agent. Further, it is advantageous to add said agent at a moderately active rate. The reaction is brought to completion by further maintaining the temperature at approxi mately C. for an additional 30 minutes.

A convenient work-up of the aforedescribed reaction consists of dilution with ice and water, followed by filtration of the crystallized product. The product may be recrystallized from or triturated with an appropriate solvent.

The requisite 3-methyl-Z-quinoxalinecarboxylic acid, alkyl esters 1,4-dioxides employed in the aforementioned reaction are most conveniently prepared by contacting 3 4 an appropriately substituted benzofuroxan and an alkyl uct formation results because of the two orientation possiacetoacetate as follows: bilities of the acetoacetate fragment in the final product.

is it co R i 2 N CH3\ c=o X x o A -e 2 N 3 N A l d .0 -K

. For exam le if one reacts a S-substituted benzofuroxan As a necessary element of the herein described process, of the forfimlaz the reaction sequences described above are effected in N the presence of a base. Such a base is of varied character. X For instance, it is meant to contemplate such bases as 0 organic amines, ammonia, alkali metal hydroxides, hy- K drides and alkoxides. Representative of such bases are l ammonia, primary amines such as n-propylamine, n-butyl- O amine, aniline, cyclohexylamine, benzylamine, p-toluidine, Wlth a l f Ha Q 2 Q2 Products, 3 ethylamine, ocwlamineg tertiary amines such as diethyh and 7-subst1tuted quinoxaline-di-N-oxide, result as shown h l aniline, N-methylpyrrolidine, N,N-d1methylpyr1m1d1ne, by t e formu ac N methylmorpholine, and 1,5 diazabicyclo-[4,3,0]-5- 1 g nonene; sodium hydroxide, potassium hydroxide, ammo- X niurn hydroxide, sodium ethoxide, potassium methoxide, and and sodium hydride. The preferred base to use is an ora \N/ -C01-R ganic amine, ammonia or alkoxide. O o

In Practice a solution or Suspension of the appropriately If the corresponding G-substituted benzofuroxan is emsubstituted benzofuroxan and the requisite acetoacetate l d as h t ti i l, th same two ibl in a reaction-inert solvent such as ethanol, N,N-dimethylproducts are formed. f id benzene, t h d f chloroform or The mixture of isomers is recovered by methods known to those skilled in the art. In many of the preparations disclosed wherein a solid, often crystalline material, separates from the reaction mixture, the solid appears to hexamethylphosphoramide is treated with an alkoxide, e.g., sodium ethoxide. It is preferable to use at least an equimolar amount of the benzofuroxan and acetoacetate, while consist predominantly of of one of the isomers. Said the amount of base may be from a catalytic to an equi- F can be Purified y repeated recrystallization from molar amount The reaction is Carried out at ambient a suitable solvent to a constant melting point. The other a isomer the one present in smaller amounts in the origitem eratures, althou h 1t ma be heated to 100 C. to

hast en product form ation, Reirction time is not critical, g gg g figg fi 22 g ig giz g 5 225;??? but will y depending on the reactivity of the Starting methods known to those skilled in the art, as for example, materials, temperature and Solvent p y d. Substantial the evaporation of the mother liquor and repeated crysyields of the desired products are isolated with reaction talization of the residue to a product of constant melting i d of 15 minutes to 24 h point. Alternatively, the reaction mixture can be extracted The requisite benzofuroxanes and acetoacetates are elther before aftFr evaporatlon to dryness either readily available or easily prepared by those Although sald mixtures may be separated by methods known to those skilled in the art for ractical reasons skilled in the art. For instance, the synthesis of variously it is advantageous to use said mhiturespas they are substituted benzofuroxans is described by Kaufman et al., lated fro the reaction Further, it is fre uently advam in Advan- Heterocyclic -a 1 (1969). Acetotageous to purify these mixtures of isomers by at least acetates are readily prepared from diketene according to one recrystallization from an appropriate solvent 01 by the general procedure f Kimel et 1 t Am Chem Soc, triturationin an appropriate solvent. Said recrystallization 65, 1995 (1943) or trrturahonthus allows the separation of the mixture of positlonal 1somers from such extraneous materials as Qu1noxalone-d1-N-ox1des result from the condensation Starting material and undesirable byproducts of benzofuroxan and substituted benzofuroxans with The identification of the isomers has not been comacetoacetic esters, such that the 2- and 3-positions of the pleted. Both isomers of a given compound, however, resulting annellated structure represent the carbonyl carexhibit the Same y of activity, -g-: as antibacterial hon and the carbon of the active methylene group of the agents or as growth, PTOmOYamS- acetoacetate Products of the instant lnventron of Formula II are I prepared by the reaction of the aforedescribed 3-halo- The substituents on the benzene moiety of the 3-methylmethyhzhuihoxahhecarhoxyhc acid, alkyl ester l 4 Z-quinoxalinecarboxylic acid ester-1,4-dioxides can vary id f Formula I with a namine of the formula R INHZ widely. For example, at least one of the following subwherein R is as previously described.

stituents can be present: hydrogen, methyl, methoxy, In practice, the 3 halomethyl 2-quinoxalinecarboxylic hl fluoro bromo d t ifl o th L Th f d acid, ester 1,4-dioxide dissolved in a reaction-inert solvent positions on the fused benzene ring of Said final products such as acetonitrile, dimethylformamide, tetrahydrofuran are the 6- or 7-positi0ns. The favored positions for suby phosphoramide, and cooled to 10-15 C.

is treated with at least two moles of the requisite amine, and as much as a ten fold excess. Instances wherein amine leading to said final products are the 5- or 6-pos1t1ons. is ammonia or methylamine said amine may be bubbled When one of said substituted benzofuroxans is condensed into the reaction media The reaction is carried out at with the requisite acetoacetate, a 6- and 7-substituted temperatures of 0-50 C. with a preferred range of 15-25" quinoXaline-di-N-oxide are produced. This multiple prod- 5 C. and for a reaction time of a few minutes to 6 hours.

stituents on the aryl ring of the starting benzofuroxans 6 A convenient work-up procedure consists of filtration useful as industrial antimicrobials, for example, in water of the precipitate, followed by a water slurry, filtration treatment, slime-control, paint preservation and wood and drying. Alternatively, the reaction mixture may be preservation as well as for topical application purposes poured into water and the resulting precipitated product as distinfectants.

filtered. For in vitro use, e.g., for topical application, it will As will be appreciated, the aforedescribed reaction lead- 5 often be convenient to compound the selected product ing to 2 substituted 2,3 dihydro 1 oxo-lH-pyrrolowith a pharmaceutically-acceptable carrier such as vege- [3,4-b]quinoxaline-4,9-dioxides may be carried out with table or mineral oil or an emollient cream. Similarly, a wide variety of amines, including alkyl-, and substituted they may be dissolved or dispensed in liquid carriers or alkylamines wherein said substituent is hydroxy, alkoxy, 1O solvents such as water, alcohol, glycols or mixtures therecycloalkyl, aryl, dialkylamino, carboalkoxy, carboxamido of or other pharmaceutically acceptable inert media, that and polyfluoroalkyl as Well as aryl-, cycloalkyl-, alkenylis, media which have no harmful effect on the active inand bibycloalkyl amines. All these compounds are congredient. For such purposes, it will generally be acceptsidered within the scope of said invention. able to employ concentrations of active ingredients of An alternate synthetic route leading the products of this from about 0.01 percent to about 10 percent by weight invention of Formula II consists of the condensation of based on total composition.

a requisite benzofuroxan and an appropriately substituted In determining the in vitro activity of an antibotic, the 3-hydroxy-3-pyrrolin-5-one as follows: sensitivity of the various micro-organisms is determined The synthetic procedure encompasses, in general, the by the commonly accepted two fold serial dilution techsame reaction conditions utilized in the preparation of the nic. Final concentrations of compound per ml. range from aforedescribed 3 methyl 2-quinoxalinecarboxylic acid, 100 mcg. in the first tube to 0.19 mcg. in the tenth tube. alkyl ester, and comprises treating an equimolar mixture The inoculum consists of 0.5 ml. of a l l0- dilution of the requisite benzofuroxan and pyrrolin-S-one in a suitof a standardized culture. Final volume in each tube or able solvent, e.g., ethanol, with a base selected from the cup in the Dis Poso tray is 1.0 ml. The tubes are incuaforementioned group. The amount of said base may be bated at 37 C. for approximately 24 hours. The medium from a catalytic to equimolar amount. The reaction is used is Witkins synthetic or Brain Heart Infusion (BHI). carried out at room temperature, although it may be The sensitivity (MICminimal inhibitory concentration) heated to approximately 100 C. to hasten product formaof the test organism is accepted as evidenced by the abtion. Reaction time will vary from about 20 minutes to sence of gross turbidity. 24 hours depending on reactivity of starting materials, Further, compounds of Formula II, described herein, solvent and reaction temperature. exhibit broad spectrum activity, that is, activity against Reaction products from both described synthetic routes both Gram-negative and Gram-positive bacteria, in conare identical in every respect when compared by melting trast to the usual Gram-negative activity of quinoxalinepoint, infrared spectroscopy, mass spectroscopy and nudi-N-oxides. Additionally, they are active in vivo and are clear magnetic resonance spectroscopy. especially useful as animal growth promotants, especially Further, this alternate procedure is employed for the for swine and poultry. preparation of those compounds within the purview of 4 When used in vivo for such purposes, these novel comthis invention related to 11 wherein the fused five mempounds can be administered orally or parenterally, e.g., bered ring is expanded to a six or seven membered by subcutaneous, intramuscular, or intravenous injecstructure as follows: tion, at a dosage of from about 1 mg./kg. to about 100 wherein X and R are as previously indicated and Z is mg./kg. of body weight. Vehicles suitable for parenteral an integer of 2 or 3. injection may be either aqueous such-as water, isotonic AS Previously indicated, the quiIloxalille'di-N-oXides 0f saline, isotonic dextrose, Ringers solution, or non-aquethe Present invention are readily adapted to therapeutic ous such as fatty oils of vegetable origin (cottonseed, use as antibacterial agents and as intermediates leading peanut n com, Sesame), di th l lf id and other to chemotherapeutic products. Iyp al member non-aqueous vehicles which will not interfere with ther- Pounds vitro 'fmubactenal agents and as apeutic efiiciency of the preparation and are nontoxic in mtlarmediates m h Include 9 the volume or proportion used (glycerol, propylene glyqumoxalmecgflljgxyh? ethig ig g i i col, sorbitol and dimethylacetamide). Additionally, com- S'bromomet y 'qumoxa mecar oxy 10 am at y es positions suitable for extemporaneous preparation of so- 1,4-dioxide. Members of interest as in vitro and in vivo antibacterial agents, as well as animal growth promotants are 2,3-dihydro-1-oxo 1H pyrrolo[3,4-b]quinoxaline-4,9-dioxide and 2-methyl-2,3-dihydro-l-oxo-lH-pyrr 10[3 4-b]quinoxaline 4 g-dioxide sorbitol, etc.; buifering agents, hyaluronrdase, local an- The valuable products of this invention of Formulae j and inofgaflic salts to aflord desirable P 1 and II are remarkably effective in treating a Wide va- 601081081 propeftles. These compounds may also-be riety of pathogenic micro-organisms. They are, therefore, bined with various pharmaceutically-acceptable inert carlutions prior to administration may advantageously be made. Such compositions may include liquid diluents, for example, propylene glycol, diethyl carbonate, glycerol,

riers including solid diluents, aqueous vehicles, nontoxic organic solvents in the form of capsules, tablets, lozenges, troches, dry mixes, suspensions, solutions, elixirs and parenteral solutions or suspensions. In general, the compounds are used in various dosage forms at concentration levels ranging from about 0.5 percent to about 90 percent by weight of the total composition.

The in vivo efficacy of the compound of Formula II of the instant invention is determined by the antibacterial activity against acute infections in mice. The acute experimental infections are produced by the intraperitoneal inoculation of standardized culture suspended in either hog gastric mucin or broth. A brief discussion of the words standardized culture would seem to be beneficial. In order to obtain reproducible results with a test compound it is necessary to control, as much as possible, the many variables that can enter into this type of test. An organism of high virulence if used in large enough numbers can make almost any drug look inactive. On the other hand, an inoculum not able to produce a measurable difference between treated and untreated groups is equally lacking in purpose.

Stock cultures of test organisms are normally maintained on slants or in liquid medium. When not routinely used they are maintained at refrigerator temperature or in a lyophilized state. When it becomes necessary to use a culture in animal protection tests the culture is suspended in a volume of saline or broth, and the density of the suspension is measured by a photoelectric colorimeter. From this stock, ten-fold dilutions are prepared. Each dilution is inoculated into a series of mice in order to determine the LD LD being the lowest concentration of organisms required to produce 100 percent deaths. For example, if it is found that a dilution of is the lowest level of organism that will produce 100 percent death, an inoculum of 10- would probably be used for the drug evaluation experiments. This means that We are using about 10 LD or ten times the minimum dose re quired to kill mice. Such a test would also include the use of control animals which receive an inoculum of 10 10- and possibly 10-*. These dilutions are included as a check on possible variation in virulence which can occur. Having previously determined, through the virulence titration, that 10- Was the maximum dilution that will kill we naturally expect these animals to die, usually Within 24 hours.

Each organism has its own standardized inoculum level. Some, such as Staphylococcus, may be used at l0 while other like Streptococcus require Weekly animal passage in order to maintain virulence.

When evaluating an antibiotic for its effectiveness after a single dose, the dose is usually administered 0.5 hour after inoculating the mice with the lethal concentration of organisms. In this type of treatment schedule surviving mice are usually held for four days after the treatment and the percent alive is calculated.

Other methods of administration of the useful products of this invention to animals include mixing with animal feeds, the preparation of feed concentrates and supplements and dilute solutions or suspensions, e.g., a 0.1 percent solution, for drinking purposes. The addition of a low level of one or more of the herein described quinoxaline-di-N-oxides to the diet of healthy animals, both ruminant and non-ruminant, such that these animals receive the product over an extended period of time, at a level of from about 0.1 mg./kg. to about 100 mg./kg. of body weight per day, especially over a major portion of their active growth period, results in an acceleration of the rate of growth and improves feed efficiency (the number of pounds of feed required to produce a pound. gain in weight). Included in these two classes of animals are poultry (chickens, ducks, turkeys), cattle, sheep, dogs, cats, swine, rats, mice, horses, goats, mules, rabbits, mink, etc. The beneficial effects in growth rate and feed efiiciency are over and above What is normally obtained with complete nutritious diets containing all the nutrients, vitamins, minerals, and other factors known to be required for the maximum healthy growth of such animals. The animals thus attain market size sooner and on less feed.

The herein described feed compositions have been found to be particularly valuable and outstanding in the case of swine. In some instances the degree of response may vary with respect to the sex of the animals. The products may, of course, be administered in one component of the feed or they may be blended uniformly throughout a mixed feed; alternatively as noted above, they may be administered in an equivalent amount via the animals water ration. It should be noted that a variety of feed components may be of use in the nutritionally balanced feeds. Any animal feed composition may be prepared to comprise the usual nutritional balance of energy, proteins, minerals and vitamins together With one or more of the quinoxaline-di-N-oxides described above. Some of the various components are commonly grains such as ground grain and grain by-products; animal protein substances, such as meat and fish by-products; vitaminaceous mixtures, e.g., vitamin A and D mixtures, riboflavin supplements and other vitamin B complexes; and bone meal, limestone and other inorganic compounds to provide minerals.

The relative proportions of the present compounds in feeds and feed concentrates may vary somewhat, depending upon the compound, the feed with which they are employed and the animal consuming the same. These substances are advantageously combined in such relative proportions with edible carriers as to provide pre-mixes or concentrates which may readily be blended with standard nutritionally balanced feeds or which may be used themselves as an adjunct to normal feedings.

In the preparation of concentrates a wide variety of carriers, including the following: soybean oil meal, corn gluten meal, cottonseed oil meal, sunflower meal, linseed oil meal, cornmeal, limestone and corncob meal can be employed. The carrier facilitates uniform distribution of the active materials in the finished feed with which the concentrate is blended. The concentrate may be surface coated, if desired, with various protenaceous materials, or edible waxes, such as zein, gelatin, microcrystalline wax and the like to provide a protective film which seals in the active ingredients. It will be appreciated that the proportions of the drug preparation in such concentrates are capable of wide variation since the amount of active materials in the finished feed may be adjusted by blending the appropriate proportion of concentrate with the feed to obtain the desired degree of supplementation. In the preparation of high potency concentrates, i.e., pre-mixes, suitable for blending by feed manufacturers to produce finished feeds or concentrates of lower potency, the drug content may range from about 0. g. to 50 g. per pound of concentrate. The high potency concentrates may be blended by the feed manufacturer with proteinaceous carriers, such as soybean oil meal, to produce concentrated supplements which are suitable for direct feeding to animals. The proportion of the drug in these supplements may vary from about 0.1 to 10 g. per pound of supplement. A particularly useful concentrate is provided by blending 2 g. of drug with 1 pound of limestone or 1 pound of limestone-soybean oil meal (1:1). Other dietary supplements, such as vitamins, minerals, etc., may be added to the concentrates in the appropriate circumstances.

The concentrates described may also be added to animal feeds to produce a nutritionally balanced, finished feed containing from about 5 to about g. of the herein described compounds per ton of finished feed. In the case of ruminants, the finished feed should contain protein, fat, fiber, carbohydrate, vitamins and minerals, each in an amount sufficient to meet the nutritional requirments of the animal for which the feed is intended. Most of these substances are present in naturally occurring feed materials, such as alfalfa hay or meal, cracked corn, whole oats, soybean oil meal, corn silage, ground corn cobs, wheat bran and dried molasses. Bone meal, limestone, iodized salt and trace minerals are frequently added to supply the necessary minerals and urea to provide additional nitrogen.

As is Well known to those skilled in the art, the types of diets are extremely variable depending upon the purpose, type of feeding operation, species, etc. Specific diets (for various purposes are listed by Morrison in the appendix of Feeds and Feeding, the Morrison Publishing Company, Clinton, Iowa, 1959.

In the case of non-ruminant animals, such as hogs, a suitable feed may contain from about 50 to 80 percent of grains, 3 to percent animal protein, 5 to 30 percent vegetable protein, 2 to 4 percent of minerals, together with supplementary vitaminaceous sources.

In practice, growth promotion is determined in swine, for instance, by the method wherein young pigs from 5-6 weeks of age and averaging 21.5 lbs. initial body weight are maintained on an ad libitum consumption of water and feed formulation consisting of ground yellow corn (58.1%), soybean meal (19.6%), alfalfa meal (2.0% dried skim milk (5.0%), dried whey (10.0%), stabilized animal fat (2.5%), limestone (0.6%), dicalcium phosphate 1.1% iodized salt (0.5% vitaminpremix PPM #5 (0.5%), quadruple delamix (0.05%), and zinc carbonate (156 g./2000 lbs. mix). The pigs are divided into groups of 32 pigs each and are held for a pre-experimental period of three days prior to starting the experiment. The quinoxaline-di-N-oxides of the present invention are added to the feed at a ratio of 50 g. of compound per ton of feed. After 28 days the efficacy of said added compounds on growth promotion is measured by a comparison of the growth in terms of weight gain of the treated animals with the untreated control group, which is arbitrarily assigned a weight gain index of 100. For example, if a compound effects a growth 23% greater than the control group (with a value of 100) it would be assigned a value of 123, etc.

The effect of a supplemented diet on the growth promotion in other species of animals using appropriate dose levels and feed formulations is assessed in an analogous manner The following examples are provided solely for the purpose of illustration and are not to be construed as limitations of this invention, many variations of which are possible without departing from the spirit or the scope thereof.

EXAMPLE I 3-bromomethyl-2-quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide To a solution of 248.0 g. (1.0 mole) of 3-methyl-2- quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide in 1400 ml. of dimethylformamide and warmed to 80 C. is added 161 g. (1.01 moles) of bromine in 100 m1. of chloroform over a period of 3 minutes. The temperature of the reac tion, which rises to 108 C., is allowed to return to 80- 85 C. where it is maintained for an additional 30 minutes. The reaction mixture is then poured into 2 liters of ice and water and stirred until crystallization is complete. The desired product is filtered, washed with water and air dried, 249 g., M.P. 108-111 C. Recrystallization from dimethylformamide-water provides the pure product, M.P. 126 C.

EXAMPLE II 3-bromomethyl-6- and 7-chloro-2-quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide To 500 ml. of dimethylformamide containing 110 g. (0.39 mole) of 3-methyl-6- and 7-chloro-2-quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide and heated to 90 C. is added over a period of 3 minutes 62 g. (0.39 mole) of bromine in 60 ml. of methylene chloride. The reaction mixture is allowed to stir at this temperature for 30 minutes after which it is added to 2.5 liters of ice and water. The resulting precipitate is filtered, washed with water and oven dried, 106 g.

EXAMPLE III Employing the procedures of Examples I and H, and starting with the appropriate starting materials, the following compounds are prepared:

N CHzBl' To 24.8 g. (0.1 mole) of 3-methyl-2-quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide contained in 100 ml. of dimethylformamide at -90 C. is added 7.8 g. (0.11 mole) of chlorine gas dissolved in 200 ml. of chloroform over a period of 23 minutes. The reaction is maintained at this temperature for an additional 35 minutes and is then poured into 1 liter of ice and water. The cold mixture is allowed to stir until the crystallization of the product is complete. The supernatant is decanted from the precipitate and the solids triturated with cold water and filtered. After air-drying the desired product is recrystallized from dimethylformamide-water.

EXAMPLE V Using the procedure of Example IV and employing the requisite starting materials, the following analogs are prepared:

0 111 j-C0aR N CHzCl Methylamine gas is bubbled into a cold -13" C.) solution of 4 litres of acetonitrile containing 250 g. (0.77 mole) of 3 bromomethyl-Z-quinoxalinecarboxylic acid, ethyl ester l,4-dioxide. After a short period of time, a voluminous precipitate starts to form. Treatment with the methylamine is continued until the precipitation of the product is complete. The solids are filtered, slurried in boiling methanol and refiltered, 203 g. A small sample is recrystallized from acetonitrile, M.P. 199-200" C.

Analysis.Calcd. for C H N O (percent): C, 57.14; H, 3.92; N, 18.18. Found (percent): C, 56.74; H, 3.84; N, 18.01.

EXAMPLE VII Employing the general procedure of Example VI, and starting with the appropriate quinoxaline-di-N-oxide and amine, the following congeners are prepared:

2,3-dihydro-1-oxo-lH-pyrrolo[3,4-b]-4,9-dioxide,

M.P. 183-184 C. 2-ethyl-2,3-dihydro-1-oxolH-pyrrolo [3,4-b] -4,9-

dioxide, M.P. 193-194 C. 2- (Z-hydroxyethyl -2,3 -dihydro-1-oxo-lH-pyrrolo- [3,4-b]-4,9-dioxide, M.P. 186-187 C. 2-(2-ethoxyethyl)-2,3-dihydro-1-oxo-lH-pyrrolo [3,4-

b]-4,9-dioxide, M.P. 159-160 C. 2-a1lyl-2,3-dihydro-l-oxo-1H-pyrrolo[3,4-b]-4,9-

dioxide, M.P. 166-167 C. 2-cyclopentyl-2,3-dihydro-l-oxo-1H-pyrrolo[3,4-b]- 4,9-dioxide, M.P. 215 C. 2-benzyl-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-b]-4,9-

dioxide, M.P. 192-493 C. 2-(2-dimethylaminoethyl)-2,3-dihydro-l-oxo-lH-pyrrolo[3,4-b]-4,9-dioxide, M.P. 105-125 C. 2-(3-hydroxypropyl)-2,3-dihydro-l-oxo-lH-pyrrolo[3,4-b]-4,9-dioxide, M.P. 188-189 C. Z-(2,3-dihydroxypropyl)-2,3-dihydro-loxo-IH-pyrrolo[3,4-b]-4,9-dioxide, M.P. 143-145 C. 2-(2-phenethyl)-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-

b]-4,9-dioxide, M.P. 174-175 C.

EXAMPLE VIII Again, employing the procedure of Example VI and starting with the appropriately substituted quinoxaline-di- N-oxide and amine, the following compounds are prepared:

H H-C3H7 H i' cgHq Cl H Cl CH Cl C H Cl i-C H CH H CH C H CH3 1'1-C3H7 CH i-CgHq OCH CH 0CH C H OCH3 I1-C3H7 OCH3 l'CgHq Br CH2 Bl C2H5 Br n-C H F CH F C2H5 F H-C3H7 F i-C3H'7 CF C H CF3 11"C3H7 CF i-C H EXAMPLE IX Starting with the appropriately substituted 3-chloromethyl-2-quinoxalinecarboxylic acid, n-propyl ester 1,4- dioxide and the requisite amine, the procedure of Example VI is again repeated to provide the following congeners:

O O 1 x N-Rl H H H CH H C H H H-C3H7 H i-C H F F C H F n'C3H7 CH CH CH C H OCH H OCH CH OCH3 n-C H7 OCH3 l-C3H7 Cl H Cl CH Cl C H Bl CH3 Br CF H CF CH v CF3 n-C3H7 EXAMPLE X 2-methyl-2,3-dihydro-1-oxo-1H-pyrrolo [3,4-b] quinoxoline 4,9-dioxide To 11.3 g. (0.1 mole) of 1-methyl-3-hydroxy-3-pyrrolin-S-one dissolved in 50 ml. of ethanol containing 1.36 g. (0.02 mole) of sodium ethoxide is added, in portions, 13.6 g. (0.1 mole) of benzofuroxan. The reaction temperature is allowed to rise to 40 C., where it is maintained with intermittent cooling during the addition. The reaction mixture is heated to reflux until the reaction is complete, and is then cooled in an ice bath. The resulting product is filtered, dried and recrystallized from dimethylformamide-water. The product is identical in every respect with that prepared in Examples VII and IX.

EXAMPLE XI Starting with the appropriately substituted reagents and utilizing the procedure of Example X the following quinoxaline-di-oxides are synthesized:

/NRi CH2).

X Z R1 X Z R 1 Calls OCH: 1 CH 3 n-CsH1 OCH 3 (33H; 2 CH3 B! 2 CH3 1 CaHu CH3 2 CH3 1 n-C H1 CH 3 CzHs 3 CH3 CF: 3 CH3 2 02H; CF: 1 02H; 2 n-CaH1 1 CHzCFs 2 (CHz)zCFa 3 CHzCFzCF;

EXAMPLE XII Employing the procedures of Examples VI and X, and utilizing the appropriate starting materials, the following analogs are prepared:

EXAMPLE XIII X R1 S. aureus E. coli 14 EXAMPLE XIV Using the previously described method for determining in vivo activity, the following compounds were tested orally against Streptococcus pyogenes at 200 and 50 5 mg./kg. and against Escherechia coli at 100, 50 and 25 mg./kg., the results being recorded as the percent animals which survived:

S. pyogenes E. coli 0 100 40 80 80 100 20 (Toxic) 100 40 0 0 0 0 0 0 60 40 40 0 40 0 0 0 0 EXAMPLE XV Employing the previously described procedure for determining growth promotion in animals, 2-methyl-2,3-dihydro 1 oxo 1H pyrrolo[3,4-b]quinoxaline-4,9-dioxide was tested at 50 g./ ton of feed in swine for a period of 28 days and provided the following results:

X N-R X H R CH Weight gain index 1 145 Percent growth over control 45 EXAMPLE XVI The efiicacy of the herein described compounds in protecting against a systemic challenge infection ofv Salmonella choleraesuis var. kunzendorf in swine is exemplified by the following experiment. Young, 6-8 week old pigs are conditioned for fourteen days in isolation rooms and maintained during the entire study on a basal ration consisting of ground yellow corn (78.4% soybean meal (15%), alfalfa meal (2%), meat bone scraps (2.5%), limestone (0.4% dicalcium phosphate (0.65% iodized salt (0.5%), vitamin pre-mix PPM #5 (0.5%), quad- 60 ruple delamix (0.05%) and zinc carbonate (7.8 g./100 lbs. mix). All the pigs, which are divided into groups of six, are inoculated on day 0 with 4 ml. (approximately 2.0x l0 organisms) of the stock inoculating suspension. Treatment with the quinoxaline-di-N-oxides of the present invention is carried out on day 0 and day 1 by intramuscular injection at 12 hour intervals at doses of 2.5 and 5 mg./kg. On day 10 the percent mortality in each group is calculated. The following results are obtained.

1 5 PREPARATION A 3-methyl-2-quinoxalinecarboxylic acid, alkyl ester 1,4- dioxides (a) 3-methyl-2-quinoxalinecarboxylic acid, ethyl ester 1,4-dioxide.To a stirred solution of 910 g. (7.0 moles) of ethyl acetoacetate in 3 liters of ethanol is added 1.4 liters of a 1 N solution of sodium ethoxide in ethanol (1.4 moles) followed by 952 g. (7.0 moles) of hemefuroxan in 30-40 g. portions. The temperature of the reaction mixture is allowed to rise to 40 and is maintained at that temperature by intermittent cooling in an ice bath. After the reaction is allowed to stir at room temperature for 2 hours, it is refluxed for an additional 16 hours and cooled in an ice bath. The precipitated product is filtered, washed with ethanol and dried, 996 g., M.P. 135-137" C.

(b) General procedure-To an ethanol solution of the requisite alkyl acetoacetate containing the basic catalyst, sodium ethoxide, is added an equimolar amount of the appropriately substituted benzofuroxan at such a rate that the temperature of the reaction mixture is easily maintained at 35-40 C. by occasional cooling with an ice bath. The reaction is brought to completion by limited heating at the reflux temperature for 6-20 hours, which is then followed by cooling in an ice bath to precipitate the final product.

The following compounds of the formula are prepared.

H CH H z s H 1'1-C3H7 H i-C H CH3 CH3 CH C H CH3 n-C H on i-c H OCH CH OCH C H OCH3 n-C3H'1 F CH F C H F. Il-CgHq F i-C H Cl CH (:1 1-1 C1 I1-C3H7 Cl i-C H'1 Br CH Brn-C H BI i-C H7 CF CH CF c n CF3 H'C3H'1 PREPARATION B 1-rnethyl-3-hydroxy-3 -pyrrolin-5-one (A) N-methyl N carbomethoxyacetylglycine ethyl ester.To a solution of 58.5 g. of N-methylglycine ethyl ester and 73.2 ml. of triethylamine in 300 ml. of tetrahydrofuran, and cooled to 40 C., is added dropwise 68.3 g. of carbomethoxyacetyl chloride. The resulting re- :action mixture is stirred at -40 C. for 20 minutes and then allowed to warm to room temperature. The solids are filtered and the filtrate evaporated to dryness. The residue is dissolved in 200 ml. of chloroform washed with '30 ml. of aqueous potassium carbonate solution. The

16 chloroform layer is separated, evaporated to dryness and the residue fractionally distilled, 88.5 g., B.P. 1081l1 C. (0.01 111111.

(B) l methyl-3-hydroxy-3-pyrrolin-4-carbomethoxy- 5-one.To 0.015 mole of sodium methoxide is 50 ml. of absolute methanol is added dropwise 21.7 g. of N-methyl- N-carbomethoxyacctylglycine ethyl ester in ml. of absolute ethanol. The resulting solution is heated to reflux for 5 minutes and is then evaporated in vacuo to dryness. The residue is dissolved in a small volume of water, washed with chloroform and the water layer acidified with 12 N hydrochloric acid. The product is extracted with chloroform and the solvent evaporated to dryness to give 15.9 g. of the desired product, M.P. 212 C., dec.

(C) 1-methyl-3-hydroxy-3-pyrrolin-5-one.--A suspension of 17.1 g. of 1-methyl-3-hydroxy-3-pyrrolin-4-carbomethoxy-S-one in 50 ml. of water is heated to reflux and maintained at that temperature until the evolution of carbon dioxide ceases. On cooling there is deposited the desired product which is filtered and dried, 10.1 g., M.P. 234 C.

The following lactams of the formula not previously reported in the literature are synthesized by the aforementioned procedure, which is essentially the method of Schmidt et al., Ann., 664, 168 (1963):

What is claimed is:

1. A method for promoting growth and improving feed efliciency of an animalwhich comprises orally or parenterally administering to said animal a dosage amount of from about 1 mg./kg. to about 100 mg./kg. body weight per day over a major portion of the animals active growth period of a compound of the formula:

wherein X is a substituent at the 6- or 7-position selected from the group consisting of hydrogen, methoxy, trifluoromethyl, bromine, chlorine and fluorine and R is hydrogen or alkyl of from 1 to 3 carbon atoms.

2. The method of claim 1 wherein said compound is administered to said animal in admixture with a nutritionally balanced animal feed, said feed containing said compound in an amount whereby the average daily ration of the animal supplies said dosage amount.

3. A method for promoting growth and improving feed efiiciency of a healthy animal which comprises orally or parenterally administering to said animal a dosage amount of from about 1 mg./kg. to about 100 nag/kg. of body Weight per day over a major portion of the animal's active growth period of a compound selected from the group consisting of 2,3 dihydro-l-oxo-1H-pyrrolo[3,4- ]q oxaline-4,9-dioxide and 2-methyl-2,3-dihydro-l-oxo-1H- pyrrolo 3 ,4-b] quinoxaline-4,9-dioxide.

4. The method of claim 3 wherein said compound is 2,3-dihydro-1-oxo 1H pyrr lo[3,4-b]quinoxaline-4,9- dioxide.

ration of the animal supplies said dosage amount.

5. The method of claim 3 wherein said compound is 2- 8. The method of claim 6 wherein said compound is 2- methyl-2,3-dihydro-1-oxo-1H-pyrro1o[3,4 bJquinoxalinemethyl-2,3-dihydro-1-oxo-1H-pyrrolo[3,4 b]quinoxa1ine- 4,9-dioxide. 4,9-dioxide.

6. The method of claim 3 wherein said compound is References Cited orally administered to in admixture a 5 P nutritionally balanced ammal feed, said feed containing said compound in an amount whereby the average daily 3,371,090 2/1968 Johnston 260-240 7. The method of claim 6 wherein said compound is ALBERT MEYERS Primary Examiner 2,3-dihydro-1-oxo 1H pyrrolo[3,4-b]quinoxaline-4,9- 10 F. E. WADDELL, Assistant Examiner dioxide.

.JUNITED. STATES ATENTIOFFI EJ 1 v CERTIFICATE OF CORRECTIQN fa e -3,773,950 Dated November 2 o,, 1973 inventofl y H y CrOnln et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: Y

Col 8,-"lir'1e 55, ?'0 g. should 'read 0.1 g. C01. 16, l nes, 0.0l5" shou1dre'ad 0.105

Signed and" sealed this 8th day of October I974.

(SEAL) Arrest:

MCCOY M. GIBSON JR. a C MARSHALL. Attesting Officer Commissioner of Patents I USCOMM-DCO376-F69 n u.s. soviamizur PRINTING umc: l9" o-ass-lu FORM PO-IOSO (10-59) 

